Neoplastic disorders such as cancer are characterized by unregulated cell proliferation. Cancer cells progress through a cycle of cellular events that include DNA replication and mitosis. A common strategy for treating such a disorder is to administer agents that inhibit either of these two events. Methotrexate, for example, is an exemplary chemotherapeutic agent for treating cancer. Methotrexate blocks DNA replication by inhibiting dihydrofolate reductases, enzymes that are required for the production of the nucleotide substrates for replication.
Eukaryotic DNA replication is a highly conserved process. Eukaryotes from fungi to mammals utilize highly related proteins to duplicate their genetic material. DNA replication can be separated into two phases: initiation and DNA synthesis. During initiation, the origin recognition complex (ORC) binds to specialized DNA sequences termed “origins.” ORC can recruit a hexameric complex of MCM (minichromosome maintenance) proteins that may function as a DNA helicase, which can unwind origins. The MCM complex in turn recruits Cdc45 (cell division cycle-45) protein and other downstream components. As a result, DNA polymerase machinery is loaded onto the origin and DNA replication can be initiated.
In normal cells, these initiation events are precisely regulated. One of the regulators is a complex formed of two proteins, Cdc7 and Dbf4. Cdc7 is a serine-threonine kinase that requires the additional subunit Dbf4 for activity. Both Cdc7 and Dbf4 are essential for survival in Saccharomyces cerevisiae. The requirement for either or both of these proteins is alleviated by bob1-1, a mutant allele of the gene CDC46/MCM5.
Dbf4 interacts with origin DNA (Dowell et al., Science, 265:1243-1246, 1999). The levels of Dbf4 protein fluctuate during the cell cycle to provide temporal regulation of Cdc7 kinase activity.
Cdc7 interacts with Orc2 in addition to Dbf4 and its kinase substrates (Hardy et al., Mol. Cell. Biol., 16:1832-1841, 1996). Human Cdc7.Dbf4 complex phosphorylates Mcm2 (Jiang et al, EMBO J., 18(20):5703-13, Oct. 15, 1999) to provide a modification which may be required for MCM complex recruitment of Cdc45. Other substrates of Cdc7·Dbf4 may include other MCM proteins, and the p 180 primase subunit.